The IL-2 receptor and related cytokine receptor systems are being studied to clarify the T cell immune response in normal, neoplastic, and immunodeficient states. Following T-cell activation by antigen, the magnitude and duration of the T-cell immune response is determined by the amount of IL-2 produced, levels of receptors expressed, and time course of each event. The IL-2 receptor contains three chains, IL-2Ra, IL-2Rb, and gc. Dr. Leonard cloned IL-2Ra in 1984, we discovered IL-2Rb in 1986, and reported in 1993 that mutation of the gc chain results in X-linked severe combined immunodeficiency (XSCID, which has a T-B+NK- phenotype) in humans. We reported in 1995 that mutations of the gc-associated kinase, JAK3, result in an autosomal recessive form of SCID indistinguishable from XSCID and in 1998 that T-B+NK+ SCID results from mutations in the IL7R gene. Based on work in our lab and others, gc was previously shown to be shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. We also previously characterized genes that were induced or repressed by IL-2, IL-4, IL-7, and IL-15, including showing the negative regulation of the IL-7 receptor alpha chain, a finding with implications in understanding how IL-2 can promote cell death, and the positive regulation of a dual specificity phosphatase, DUSP5, that negatively regulates IL-2-mediated activation of ERK kinases. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation being important for host defense to viruses and other intracelllular pathogens, Th2 differentiation being vital in allergic disorders and related to helminths, and Th17 differentiation being vital in a range of inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and reported that IL-2 regulates expression of the IL-4 receptor in a STAT5-dependent manner and critically controls priming of cells for Th2 differentiation. Moreover, using genome-wide Illumina-based ChIP-Seq (chromatin immunoprecipitation coupled to DNA sequencing) analysis, we had discovered broad regulation of Th2 differentiation via STAT5A and STAT5B, substantially extending earlier studies focused on STAT5A. Moreover, we had discovered that IL-2-mediated IL-4Ra induction was critical in priming cells for Th2 differentiation. In the prior year, we substantially extended these findings by showing that IL-2 via STAT5 induces expression of IL-12Rb1 and IL-12Rb2 and that the induction of IL-12Rb2 is critical for Th1 differentiation and we defined the mechanism of regulation of IL-12Rb2. Additionally, we showed that IL-2 via STAT5 also regulates the T box protein, T-bet. Interestingly, in contrast to the induction of IL-12R proteins, IL-2 inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. Consistent with the ability of Tbx21 to inhibit Th17 differentiation, expression of Tbx21 in Th17 cells resulted in increased IFNg but decreased expression of IL-17A. These results indicated a very broad effect of IL-2 via STAT5 on T helper cell differentiation. In the current review year, we have continued to study the role of IL-2 in Th differentiation, extending findings to Th9 differentiation. We also reported major discoveries related to the role of STAT5 tetramerization in vivo. In addition to forming dimers, a number of STAT proteins can form tetramers via N-terminal region (N-domain)-mediated oligomerization of STAT dimers. Using the previously defined three dimensional structural information for the N-domain of STAT1 and STAT4, we predicted the key residues in STAT5A and STAT5B that are required for N-domain-mediated oligomerization, and we confirmed their importance by mutagenesis coupled to electrophoretic mobility shift assays. We then made single and double knockin mice for STAT5A and STAT5B to generate animals that could form STAT5 dimers but not tetramers. Using Affymetrix arrays and RNA-Seq methodology, we defined the role of STAT5 tetramerization for gene expression. Using ChIP-Seq, we also defined the consensus motifs that were required for STAT5 dimer versus tetramer formation. We also coupled the ChIP-Seq and RNA-Seq data to define tetramer-regulated genes and to define key binding sites. We found that a key set of genes required STAT5 tetramers for normal expression. We also found that STAT5 tetramers were essential for normal T cell expansion/proliferation as well as survival, and demonstrated in an inflammatory bowel disease model that STAT5 tetramers are needed for normal development of regulatory T cells. These findings are the first to reveal key actions for STAT tetramers in vivo. Moreover, they have implications related to the development of new agents that selectively inhibit tetramer function. During this year, we also have collaborated with Dr. K. Christopher Garcia at Stanford, studying the actions of wild type IL-2 versus novel IL-2 variants, a project with potential clinical ramifications. These studies in part use the pmel-1 T cell receptor transgenic model of adoptive immunotherapy for cancer in collaboration with Dr. Nicholas Restifo, NCI. We have also collaborated with Dr. Garcia on a project in which he compared the three dimensional structure of IL-2 complexed to its receptors to that of IL-15 bound to its receptor. These studies have provided key mechanistic and structural insights into the functional differences between IL-2 and IL-15, which are highly related and share IL-2Rbeta and gc as receptor components but nevertheless possess distinctive biological functions. Although IL-2 primarily signals via cis-signaling and IL-15 via trans-signaling, these cytokines have essentially identical activation of STAT, PI3K/Akt, and Ras/MAPK signaling pathways. Moreover, gene expression profiles are very similar, although not identical. Thus, these cytokines have almost indistinguishable signaling properties despite different biological responses. This study has substantially elucidated structural and mechanistic aspects of IL-2 and IL-15 signaling. We previously reported the generation of IL-2/IL-21 dual reporter BAC transgenic mice. These mice allow one to observed through surrogate fluorescent markers the relative induction of these cytokines in different cells. Using these mice, we previously demonstrated a key role for IL-21 in a model mouse system for experimental autoimmune uveitis, and we had demonstrated the existence of IL-2, IL-21, and IL-2/IL-21 double expressing cells in the retina, consistent with a role for this cytokine in the disease process. We are continuing to develop studies with these valuable animals, with the intent of learning more about the roles of IL-2 and IL-21 in vivo. Overall, these studies help to improve our understanding of signaling by gc family cytokines. These findings clarify basic molecular mechanisms that are relevant to normal and pathological immune cell function, including allergy, autoimmunity, and cancer.